Double Immunohistochemical Labelling of PRAME and Melan A in Slow Mohs Biopsy Margin Assessment of Lentigo Maligna and Lentigo Maligna Melanoma.

Introduction: Lentigo maligna (LM) and lentigo maligna melanoma (LMM) predominantly affect the head and neck areas in elderly patients, presenting as challenging ill-defined pigmented lesions with indistinct borders. Surgical margin determination for complete removal remains intricate due to these characteristics. Morphological examination of surgical margins is the key form of determining successful treatment in LM/LMM and underpin the greater margin control provided through the Slow Mohs micrographic surgery (SMMS) approach. Recent assessments have explored the use of immunohistochemistry (IHC) markers, such as Preferentially Expressed Antigen in Melanoma (PRAME), to aid in LM/LMM and margin evaluation, leveraging the selectivity of PRAME labelling in malignant melanocytic neoplasms. Methods: A Novel double-labelling (DL) method incorporating both PRAME and MelanA IHC was employed to further maximise the clinical applicability of PRAME in the assessment of LM/LMM in SMMS biopsies. The evaluation involved 51 samples, comparing the results of the novel DL with respective single-labelling (SL) IHC slides. Results: The findings demonstrated a significant agreement of 96.1% between the DL method and SL slides across the tested samples. The benchmark PRAME SL exhibited a sensitivity of 91.3% in the SMMS specimens and 67.9% in histologically confirmed positive margins. Discussion: This study highlights the utility of PRAME IHC and by extension PRAME DL as an adjunctive tool in the assessment of melanocytic tumours within staged excision margins in SMMS samples.

Evaluation of a New Mordant Based Haematoxylin Dye (Haematoxylin X) for Use in Clinical Pathology.

Recently, St John's Dermatopathology Laboratory and CellPath Ltd have developed a new patented haematoxylin dye (Haematoxylin X) that utilises a chromium-based mordant (Chromium Sulphate). In this study, the performance of this new haematoxylin (Haematoxylin X) was compared against some commonly utilised alum-based haematoxylins (Carazzi's, Harris' and Mayer's) when used as a part of formalin-fixed paraffin embedded (FFPE) tissue, special stains, immunohistochemical counterstaining and frozen section (Mohs procedure) staining procedures. FFPE sections of different tissue types and frozen skin tissues were sectioned and stained with each haematoxylin subtype to allow for a direct comparison of staining quality. The slides were independently evaluated microscopically by two assessors. A combined score was generated to determine the sensitivity (defined as the intensity of haematoxylin staining being too weak or too strong and the colour of the haematoxylin staining not being blue/black) and specificity (defined as the presence of haematoxylin background staining, uneven staining, and staining deposits) for each of the four haematoxylin subtypes. The scoring criteria were based on the UKNEQAS Cellular pathology techniques assessment criteria. In FFPE tissue, the results for specificity identified Harris haematoxylin scoring the highest (91.2%) followed by Haematoxylin X (88.0%) and Mayer's (87.0%). The sensitivity scores again identified Harris haematoxylin as scoring the highest (95.1%) followed by Haematoxylin X (90.0%) and Mayer's (88.0%). In frozen tissue, the results for specificity identified Haematoxylin X as scoring the highest (85.5%) followed by Carazzi's (80.7%) and Harris' (77.4%). The sensitivity scores again identified Haematoxylin X as scoring the highest (86.8%) followed by Carazzi's (82.0%) and Harris' (81.0%). The results achieved with all four haematoxylins showed a high degree of comparability, with Harris' haematoxylin scoring high scores overall compared to the other four when assessing FFPE sections. This may have been due to familiarity with the use of Harris' haematoxylin in-house. There was also evidence of more pronounced staining of extracellular mucin proteins with Haematoxylin X compared to the other alum haematoxylins that were assessed. Haematoxylin X scored highest when used in frozen section staining. In addition, Haematoxylin X has a potential applications for use in IHC and special stains procedures as a counterstain.

Gout With Associated Cutaneous AA Amyloidosis: A Case Report and Review of the Literature.

Gout with associated AA amyloidosis is an unusual finding. This form of amyloid is associated with chronic inflammatory changes often associated with amyloid deposits in the urine, as well as tissue involvement, and organ enlargement in some cases. The large majority of cases in the literature to date refer to gout with AA amyloid within the kidney. However, this is not exclusive, with reports in the liver, gastrointestinal tract, adrenal glands rectum, skin, and subcutaneous fat. The pathophysiological association between these two disease processes is open to debate. The employment of specific anti-inflammatory treatments is believed to have an impact on reducing the incidence of AA amyloidosis in some gout cases-notably the use of colchicine in cases of clinically defined gout attacks. However, this is by no means a universal finding. Here we report on a cutaneous case of gout with AA amyloidosis in a 73-year-old man Included in this case study is a review of the other 16 cases reported within the literature in an attempt to clarify the associated pathophysiological process between these two diseases and the anti-inflammatory treatment regimens employed which may impact the occurrence of AA amyloidosis.

Evaluation of different haematoxylin stain subtypes for the optimal microscopic interpretation of cutaneous malignancy in Mohs frozen section histological procedure.

BACKGROUND: The Mohs technique employs mainly H&E-stained frozen sections for surgical margin assessment of cutaneous excisions, utilising microscopic evaluation of the complete, circumferential, peripheral and deep margins. This study aimed to determine which mordant based haematoxylin (Ehrlich's, Cole's, Mayer's, Gill's I, Gill's II, Gill's III, Weigert's, Harris' or Carazzi's) produced the optimal morphological clarity of staining for the identification of cellular and tissue morphology of cutaneous basal cell carcinoma (BCC). MATERIAL AND METHODS: In total, 100 anonymised patient cases were selected, sectioned and stained with each haematoxylin subtype. The slides were independently evaluated microscopically by two assessors. A combined score was generated to determine the sensitivity (defined as the intensity of haematoxylin staining being too weak or too strong and the colour appearance of the haematoxylin not being blue/black) and specificity (defined as the appearance of background staining with haematoxylin, uneven staining and staining deposits) for each of the nine haematoxylin subtypes. The scoring criteria were based on the UKNEQAS CPT Mohs procedure assessment criteria. RESULTS: The scores generated for specificity identified Carazzi's haematoxylin as best performing (99.2%) followed by Gill's III (98.4%), Ehrlich's (98.2%) and Harris' (85.0%). The sensitivity score again identified Carazzi's as producing the best result (85.0%) followed by Weigert's (83.4%), Ehrlich's (81.6%) and Gill's III (80.4%). DISCUSSION: Carazzi's haematoxylin is the most optimal staining dye for the identification of BCC tumour for use as part of the Mohs micrographic surgery procedure.

British Journal of Biomedical Science in 2020. What have we learned?

Each year the British Journal of Biomedical Science publishes a 'What have we learned' editorial designed to introduce readers within the major disciplines of laboratory medicine to developments outside their immediate area. In addition it is designed to inform a wider readership of the advances in the diagnosis and treatment of disease. To this end, in 2020 the journal published 39 articles covering the disciplines within Biomedical Science in the 4 issues comprising volume 77. These included a review of COVID-19 in this issue, 27 original articles, 6 Biomedical Science 'In Brief' and 4 case histories. 27 of the articles involved molecular techniques, with one of these comparing results with a mass spectrometry based method. The preponderance of molecular genetic studies gives us a good idea of the likely future direction of the disciplines.

Semi-automated standardisation of melanin bleaching procedures of heavily pigmented melanocytic lesions for immunohistochemical analysis on an automated platform.

Background: The diagnosis of heavily pigmented melanocytic lesions is problematic. This is often compounded by lack of visibility of nuclear detail of tumour cells due to physical masking by melanin pigment. Similarly, there can be colour merging of chromogenic final reaction products with melanin, making an evidence of antigenic localisation problematic. There are a number of melanin bleaching techniques available for immunohistochemical assessments.Material and methods: All methods to date have involved the bleaching of melanin as a manually performed primary step before loading subsequently bleached slides onto automated immunohistochemical platforms. Here we define a semi-automated bleaching procedure that allows full integration on one of the most widely employed automated IHC staining platforms (Roche Ventana BenchMark Ultra). The bleaching protocol was defined on the BenchMark Ultra and involved the assessment of 24 histological cases of heavily pigmented malignant melanoma lesions (13 cutaneous and 11 metastatic) routinely fixed processed and paraffin wax embedded.Results: Completion of the bleaching was assessed on H&E preparations performed following the semi-automated bleaching step and employing the Roche Ventana BenchMark Ultra machine for 60 min at 42°C. Complete immunohistochemical staining was achieved on the automated platform within 5-6 h including the bleaching step. Results were consistent across all tissue evaluated.Discussion: This data provides evidence that the hydrogen peroxide bleaching procedure can be adapted for integration on one of the most widely employed automated IHC staining platforms and as a result, improve the efficiency and reproducibility of the technique.

Immunohistochemical detection of V600E BRAF mutation is a useful primary screening tool for malignant melanoma.

BACKGROUND: We compared the use of an immunohistochemical (IHC) method using a monoclonal antibody to BRAF V600E (which detects the main BRAF mutation) with existing DNA probe screening in tissue samples from 71 patients with malignant melanoma. MATERIALS AND METHODS: Paraffin blocks were cut to provide consecutive slides for haematoxylin and eosin staining, and for known positive micro-array DNA control material. IHC was performed by the Optiview detection system. All slides were scored independently by the clinical lead and the laboratory lead using a positive/negative system. RESULTS: The DNA method found 26 samples to be positive, the IHC found 21 to be positive, giving a sensitivity value for IHC of 80.8%. However, all of the 45 samples found to be negative by DNA were also negative by IHC, giving a specificity of 100%. There were 66 instances of full agreement, giving a concordance of 93%. Together, these data give a kappa statistic of 0.843, indicating very good agreement. CONCLUSION: The data reveal a very close link between the two methods, supporting the use of the V600E as a primary screen for BRAF mutations in malignant melanoma. Samples found to be negative by this method may be retested by the DNA probe method. IHC detection conserves patient DNA from tumour blocks as only one section is required to perform the assay. The V600E antibody method is considerably cheaper and faster than the DNA probe assay, with a turn-around time of 24-48 hours, enabling more rapid clinical management.

Use of a novel 1-hour protocol for rapid frozen section immunocytochemistry, in a case of squamous cell carcinoma treated with Mohs micrographic surgery.

For squamous cell carcinoma (SCC) treated using Mohs micrographic surgery (MMS), interpretation of haematoxylin and eosin-stained frozen sections can be challenging. In these situations, ancillary use of immunostaining is a useful tool for the Mohs surgeon. However, use of immunostaining in MMS laboratories is limited, mainly because current manual immunostaining platforms are subject to operator error, and automated immunostaining, albeit accurate, is too slow for inclusion in MMS. In this report, we describe a novel 1-hour protocol for rapid frozen section immunocytochemistry, using the pancytokeratin markers AE1/AE3. This protocol has been specifically designed to integrate the speed of manual techniques and the accuracy of automated platforms, making it a valuable addition to the MMS laboratory. We propose that in selected or histologically challenging cases, there is a role for the use of this novel protocol, allowing the Mohs surgeon to more confidently declare tumour clearance, thus preventing further unnecessary surgery and preserving healthy tissue.

New embedding and staining systems PrestoCHILL and Presto stainer for application in the advancement of Mohs micrographic surgery.

BACKGROUND: Mohs micrographic surgery (MMS) involves evaluation of frozen tissue sections to determine complete circumferential and deep tissue margin clearance of skin tumours. PrestoCHILL and Presto stainer devices are two new innovative tools which bring benefits of automation, speed and efficiency to the preparation of frozen section analysis in MMS. The devices were assessed at Viapath's Tissue Science Mohs laboratory at Guy's Cancer Centre. MATERIAL AND METHODS: A total of 279 samples from 10 anatomically different facial sites. These included nose (95), lip (24), forehead (47), cheek (25), eyelids (34), temple (9), chin (15), ear (17), scalp (6) and neck (7). These were analysed using both devices simultaneously. RESULTS: The PrestoCHILL device was measured for accuracy of tissue orientation by determining how many of the cases examined microscopically had complete margin and full epidermis preservation. The precision and reproducibility of the Presto stainer was evaluated by the consistency of achieving ideal standards of staining quality as defined by the department's internal quality control check, on stained sections examined and evaluated microscopically. The mean (standard deviation) score for accuracy for the PrestoCHILL across all tissue facial sites was 93.5 (11)%; the mean (standard deviation) score for precision/reproducibility of the Presto stainer was 96.5 (11)% (both p < 0.05). CONCLUSION: The devices combined offer an assured accuracy and precision performance, which is reproducible across all facial tissue types examined. The devices represent a key step forward in the introduction of improved automated embedding and staining procedures within MMS.

A multicentre study of the precision and accuracy of the TruSlice and TruSlice Digital histological dissection devices.

BACKGROUND: Five key factors enabling a good surgical grossing technique include a flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. TruSlice and TruSlice Digital are new innovative tools based on a guillotine configuration. The TruSlice has plastic inserts whilst the TruSlice Digital has an electronic micrometre attached: both features enable these dissection factors to be controlled. The devices were assessed in five hospitals in the UK. MATERIAL AND METHODS: A total of 267 fixed tissue samples from 23 tissue types were analysed, principally the breast (n = 32) skin (30), rectum (28), colon (27) and cervix (17). Precision and accuracy were evaluated by measuring the defined thickness, and the consistency of achieving the defined thickness of tissue samples taken respectively. Both parameters were expressed as a total percentage of compliance for the cohort of samples accessed. RESULTS: Overall, the mean (standard deviation) score for precision was 81 (11) % whilst the accuracy score was 82 (11) % (both p < 0.05, chi-squared test), although this varied with type of tissue. Accuracy and precision were strongly correlated (rp = 0.83, p < 0.001). CONCLUSION: The TruSlice Digital devices offer an assured precision and accuracy performance which is reproducible across an assortment of tissue types. The use of a micrometre to set tissue slice thickness is innovative and should comply with laboratory accreditation requirements, alleviating concerns of how to tackle issues such as the 'measurement of uncertainty' at the grossing bench.

Development of new and accurate measurement devices (TruSlice and TruSlice Digital) for use in histological dissection: an attempt to improve specimen dissection precision.

Histological dissection of human tissue has relied on conventional procedures, which have largely remained unchanged for decades. Practices to determine measurement parameters employed in these procedures have largely relied on the use of rulers and weighing scales. It is well documented in the scientific literature that both fixation and processing of tissue can significantly affect the viability of the of tissue sections both for tinctorial and immunocytochemical investigations. Both of these factors can be compounded in their negative effects by inappropriate sampling of tissue at histological cut up. There are five key factors to ensure good surgical grossing technique, flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. Meeting these factors implies the devices are fit for purpose. Here we describe an innovative approach to designing cut up devices to improve accuracy and precision, which take these five key requirements into consideration. The devices showed accuracy and precision, enabling tissue slices to be produced in a uniformly perpendicular fashion to within 2 mm in thickness and to enable consistency and reproducibility of performance across a series of tissue types. The application of a digital rule on one of these devices ensures accuracy and also enables quality control issues to be clearly assessed. As cellular pathology laboratories conform to ever increasing standards of compliance and performance in practice, the advent of assured precision and accuracy at cut up is awaited. Recommendations from accreditation bodies such as the United Kingdom Accreditation Service (UKAS) continue to push for improvements in this area of histological investigation. These newly designed devices may give the answers to these requirements and provide the impetus for a new generation of innovative equipment for histological dissection.

Pan-cytokeratin markers for rapid frozen section immunocytochemistry from head and facial Mohs cases of basal cell carcinoma: a comparison and evaluation to determine the marker of choice.

The application of immunocytochemistry in the field of Mohs micrographic surgery (MMS) is well established. This study evaluates the use of pan-cytokeratins (AE1/AE3, MNF116 and AE1/AE3+PCK26) in the assessment of basal cell carcinoma (BCC) on frozen tissue debulk specimens. Fifty-five cases of BCC, all from head and facial sites, were assessed in the study. In addition to staining all cases for the three cytokeratin antibodies under investigation, sections were also stained with haematoxylin and eosin (H&E) to demonstrate tumour architecture and morphology. All sections for immunocytochemistry were stained on a Roche Ventana BenchMark Ultra automated platform employing a rapid frozen section protocol. Results were assessed based on the intensity of staining of keratinocytes (scale: 0-100%), as well as sensitivity of staining determined by the total percentage of keratinocytes stained within the tissue section. AE1/AE3 demonstrated the most consistent staining both in terms of intensity of staining and sensitivity, with a mean of 99.1% and 99.9%, respectively. AE1/AE3+PCK26 average results indicated scores of 70.6% for intensity and 87.2% for sensitivity, with MNF116 scoring 92.9% for intensity but only 57.3% for sensitivity. The data indicate that AE1/AE3 is the best pan-cytokeratin antibody to use in the assessment of BCC in MMS. The use of cytokeratin immunocytochemistry is justified in morphologically complex cases of BCC, or in cases where dense inflammatory infiltrate surrounding any suspicious cells make identification of small numbers of tumour cells difficult to determine with just an H&E stain. The significant rationale is that cytokeratin staining is a valuable adjunct in the study of tumour cell assessment in cases of MMS for BCC. In addition, the use of anti-AE1/AE3 cytokeratin antibodies provides the most consistent staining results for such cases.

Dermatofibrosarcoma protuberans: dealing with slow Mohs procedures employing formalin-fixed, paraffin wax-embedded tissue in a busy diagnostic laboratory.

Dermatofibrosarcoma protuberans (DFSP) is a relatively uncommon tumour that arises in the dermis and underlying soft tissue. Surgical removal is the preferred treatment, with relatively wide clearance margins of 3 cm or more. Slow Mohs procedures are often employed successfully to treat patients with such tumours. Slow Mohs procedures offer the benefit of improved cure rates and maximal tissue conservation. However, dealing with such tissue successfully presents the laboratory with a host of technical problems. This report advocates a set protocol to follow for slow Mohs, based on the experience acquired from dealing with 37 cases of DFSP over a 12-year period. The report establishes the benefits of slow Mohs paraffin wax-embedded tissue over frozen sections in terms of improved morphology, tissue preservation and immunocytochemical labelling with anti-CD34.

The feasibility of in vivo quantification of bone-fluorine in humans by delayed neutron activation analysis: a pilot study.

Fluorine (F) plays an important role in dental health and bone formation. Many studies have shown that excess fluoride (F(-)) can result in dental or skeletal fluorosis, while other studies have indicated that a proper dosage of fluoride may have a protective effect on bone fracture incidence. Fluorine is stored almost completely in the skeleton making bone an ideal site for measurement to assess long-term exposure. This paper outlines a feasibility study of a technique to measure bone-fluorine non-invasively in the human hand using in vivo neutron activation analysis (IVNAA) via the (19)F(n,γ)(20)F reaction. Irradiations were performed using the Tandetron accelerator at McMaster University. Eight NaI(Tl) detectors arranged in a 4π geometry were employed for delayed counting of the emitted 1.63 MeV gamma ray. The short 11 s half-life of (20)F presents a difficult and unique practical challenge in terms of patient irradiation and subsequent detection. We have employed two simultaneous timing methods to determine the fluorine sensitivity by eliminating the interference of the 1.64 MeV gamma ray from the (37)Cl(n,γ)(38)Cl reaction. The timing method consisted of three counting periods: an initial 30 s (sum of three 10 s periods) count period for F, followed by a 120 s decay period, and a subsequent 300 s count period to obtain information pertaining to Ca and Cl. The phantom minimum detectable limit (M(DL)) determined by this method was 0.96 mg F/g Ca. The M(DL) was improved by dividing the initial timing period into three equal segments (10 s each) and combining the results using inverse variance weighting. This resulted in a phantom M(DL) of 0.66 mg F/g Ca. These detection limits are comparable to ex vivo results for various bones in the adult skeleton reported in the literature. Dosimetry was performed for these irradiation conditions. The equivalent dose for each phantom measurement was determined to be 30 mSv. The effective dose was however low, 35 µSv, which is comparable to other clinical diagnostic tools. The M(DL), relatively low radiation dose and non-invasiveness indicate the suitability of this method for routine in vivo analysis of bone-fluorine content. This prompted us to perform a trial study in human subjects. A preliminary human study on 34 participants was completed, with 33 of the 34 measurements proving to be successful. The in vivo M(DL) based on the improved timing method was determined to be 0.69 mg F/g Ca for the 33 successful human measurements. In our opinion, this technique has been demonstrated to be a suitable method for in vivo assessment of fluorine bone-burden.

Comparison of ex vivo optical coherence tomography with conventional frozen-section histology for visualizing basal cell carcinoma during Mohs micrographic surgery.

BACKGROUND: Mohs micrographic surgery offers high cure rates of nonmelanoma skin cancers with optimal sparing of normal tissue. However, it is generally more time-consuming and labour-intensive than traditional surgery. Optical coherence tomography (OCT) is an emergent technology that has the potential to diagnose basal cell carcinoma (BCC) in vivo. OBJECTIVE: To compare the efficiency and accuracy of ex vivo OCT with frozen-section histology for identifying BCC in Mohs surgery. METHODS: Thirty-eight patients were enrolled. After the stages were taken, images were captured with an OCT microscope and subsequently processed for standard frozen sections. RESULTS: In total, 75 sections were scanned and the mean time to produce one OCT image was 7 min. In four of 26 positive haematoxylin-eosin sections and 23 of 49 negative sections, there was a good correlation with OCT images. The sensitivity and specificity were 19% and 56%, respectively. CONCLUSIONS: It is possible to identify BCC with ex vivo OCT and this is more rapidly obtained than with haematoxylin-eosin frozen sections. However, tumour visualization in OCT was disappointing. Practical benefit may be obtained by optimizing this technology and combining it with other new diagnostic tools.

Investigation into a new softening agent for use on formalin-fixed, paraffin wax-embedded tissue.

The use of tissue softening agents to improve microtomy of keratotic tissues is employed widely. Many of these softeners contain hazardous constituents such as phenol. In this study, the use of non-ionic surfactants or non-toxic ingredients are investigated with the aim of creating a new softening agent. The new agent should be more effective in facilitating the sectioning of hardened tissue while reducing toxicity and complications associated with sectioning hard tissue compared to a commercially available phenol-based formulation. Four formulations are compared against the commercial product for their capability to section routinely processed paraffin-embedded tissue under standard operating procedure parameters. The trial formulations were shown to be fast acting and enabled improved serial sectioning of hard keratotic tissue in nearly all the cases tested. There was no evidence of adverse staining using either tinctorial or immunohistochemical methods. The new formulations had advantages over the commercially available solutions, improving on the number and quality of sections attainable from the tissue blocks, as well as offering a composition less toxic than phenol-based products.